EST-SSR Markers Research Articles

Comparative analysis of genetic diversity and population structure of wild barley (Hordeum vulgare subsp. spontaneum) in Western Iran using SSR and EST-SSR markers

Wild barley (Hordeum vulgare subsp. spontaneum), the progenitor of cultivated barley (Hordeum vulgare subsp. vulgare), is crucial for enhancing genetic diversity in breeding programs. The genetic diversity and structure of wild barley populations in western Iran are not well understood. This study evaluated 114 wild barley genotypes from four provinces in western Iran using SSR and EST-SSR markers. SSR markers revealed a considerable number of alleles, with an average of 2.7 per marker, while EST-SSR markers exhibited a slightly higher mean allele count of 2.8 per marker. Most genetic variation occurred within populations (97% for SSR and 98% for EST-SSR), with low differentiation among populations (Fst = 0.033 and 0.026). Various diversity indices were calculated, with SCSSR04163 showing the highest values. Jaccard coefficients revealed significant genetic distance among genotypes, especially between populations from different provinces. Cluster analysis categorized the genotypes into distinct groups based on SSR and EST-SSR markers. The Mantel test showed weak correlation (r = 0.10048, p = 0.9738) between the two dendrograms. Population structure analysis identified three optimal clusters (K = 3). This study demonstrates the utility of SSR and EST-SSR markers in understanding the genetic diversity and structure of wild barley, providing valuable insights for conservation and improvement.

Genetic diversity, core collection construction and genotype–environment associations of Eucalyptus cloeziana germplasm based on EST-SSR markers

Genetic diversity, core collection construction and genotype–environment associations of Eucalyptus cloeziana germplasm based on EST-SSR markers

Transcriptome sequencing on different ages of Saraca asoca bark: Insights from tannin biosynthetic pathways and EST-SSR marker design.

Transcriptome sequencing on different ages of Saraca asoca bark: Insights from tannin biosynthetic pathways and EST-SSR marker design.

Tissue-specific transcriptomic analysis reveals the molecular mechanisms responsive to cold stress in Poa crymophila, and development of EST-SSR markers linked to cold tolerance candidate genes

BackgroundPoa crymophila is a perennial, cold-tolerant, native grass species, widely distributed in the Qinghai-Tibet Plateau. However, the tissue-specific regulatory mechanisms and key regulatory genes underlying its cold tolerance remain poorly characterized. Therefore, in this study, based on the screening and evaluation of cold tolerance of four Poa species, the cold tolerance mechanism of P. crymophila’s roots, stems, and leaves and its cold tolerance candidate genes were investigated through physiological and transcriptomic analyses.ResultsResults of the present study suggested that the cold tolerance of the four Poa species was in the following order: P. crymophila > P. botryoides > P. pratensis var. anceps > P. pratensis. Cold stress significantly changed the physiological characteristics of roots, stems, and leaves of P. crymophila in this study. In addition, the transcriptome results showed that 4434, 8793, and 14,942 differentially expressed genes (DEGs) were identified in roots, stems, and leaves, respectively; however, 464 DEGs were commonly identified in these three tissues. KEGG enrichment analysis showed that these DEGs were mainly enriched in the phenylpropanoid biosynthesis pathway (roots), photosynthesis pathway (stems and leaves), circadian rhythm-plant pathway (stems and leaves), starch and sucrose metabolism pathway (roots, stems, and leaves), and galactose metabolism pathway (roots, stems, and leaves). A total of 392 candidate genes involved in Ca2+ signaling, ROS scavenging system, hormones, circadian clock, photosynthesis, and transcription factors (TFs) were identified in P. crymophila. Weighted gene co-expression network analysis (WGCNA) identified nine hub genes that may be involved in P. crymophila cold response. A total of 200 candidate gene-based EST-SSRs were developed and characterized. Twenty-nine polymorphic EST-SSRs primers were finally used to study genetic diversity of 40 individuals from four Poa species with different cold tolerance characteristics. UPGMA cluster and STRUCTURE analysis showed that the 40 Poa individuals were clustered into three major groups, individual plant with similar cold tolerance tended to group together. Notably, markers P37 (PcGA2ox3) and P148 (PcERF013) could distinguish P. crymophila from P. pratensis var. anceps, P. pratensis, and P. botryoides.ConclusionsThis study provides new insights into the molecular mechanisms underlying the cold tolerance of P. crymophila, and also lays a foundation for molecular marker-assisted selection for cold tolerance improvement in Poa species.

Genetic diversity of cultivated Nigella sativa L. germplasm based on EST-SSR markers and agro-morphological traits

Genetic diversity of cultivated Nigella sativa L. germplasm based on EST-SSR markers and agro-morphological traits

Genetic insights into diversity and population structure of ashwagandha (Withania somnifera (L.) Dunal) using EST-SSR, ISSR and SSR markers: Implications for enhancing agricultural and industrial value

Genetic insights into diversity and population structure of ashwagandha (Withania somnifera (L.) Dunal) using EST-SSR, ISSR and SSR markers: Implications for enhancing agricultural and industrial value

Population Genetics of Haliotis discus hannai in China Inferred Through EST-SSR Markers.

The Pacific abalone Haliotis discus hannai originated in cold waters and is an economically important aquaculture shellfish in China. Our goal was to clarify the current status of the genetic structure of Pacific abalone in China. In this study, eighteen polymorphic EST-SSR loci were successfully developed based on the hemolymph transcriptome data of Pacific abalone, and thirteen highly polymorphic EST-SSR loci were selected for the genetic variation analysis of the six populations collected. The results showed that the average number of observed alleles was 8.0769 (RC)-11.3848 (DQ) in each population. The number of observed alleles in the DQ, NH, and TJ populations was significantly higher than that in the RC population. The cultivated population outside the Changshan Islands has experienced a 22.79% reduction in allele diversity compared to the wild population of DQ. The pairwise Fst values and analysis of molecular variance (AMOVA) revealed significant population differentiation among all populations except DQ and NH populations, with RC and ZZ cultured populations exhibiting the largest population differentiation (Fst = 0.1334). The phylogenetic tree and structural analysis divided the six populations into two groups (group 1: NH, DQ, and ZZ; group 2: DL, TJ, and RC), and there was no relationship between geographical distance and genetic distance. These results may reflect the large-scale culture from different populations in China and the exchange of juveniles between hatcheries. Different breeding conditions have led to a higher degree of genetic differentiation between the RC and ZZ populations. This study enables a better understanding of the genetic diversity and structure of current Pacific abalone populations.

Exploitation of novel drought responsive EST-SSR markers in tetraploid cotton (Gossypium hirsutum L.)

Exploitation of novel drought responsive EST-SSR markers in tetraploid cotton (Gossypium hirsutum L.)

Genetic Diversity and Population Structure Analysis of Pinus elliottii Germplasm Resources in Jiangxi Province.

This study aimed to compare and assess the genetic diversity and trends among the introduced family provenance, first-cycle superior trees breeding provenance, and improved-generation superior trees breeding provenance of Pinus elliottii using EST-SSR markers. The goal was to provide a foundation for advanced genetic improvement and sustainable utilization of P. elliottii in Jiangxi Province. A total of 417 individuals were analyzed for their genetic diversity and population structure using 19 pairs of SSR markers. The analysis identified 103 alleles across all the samples, with an average of 5.421 alleles per locus. Compared to other coniferous species, P. elliottii exhibited a moderate to high level of genetic diversity (I = 0.862, He = 0.457). Analysis of the molecular variance (AMOVA) revealed that 97.90% of the genetic variation occurred within provenances, consistent with a low genetic differentiation coefficient (Fst = 0.016 < 0.05) and high gene flow (Nm = 15.715) among provenances. In addition, analysis using STRUCTURE v. 2.3.4 software divided the 417 germplasm samples into two distinct groups, corroborating the results of the principal coordinates analysis (PCoA) and the unweighted pair group method with arithmetic (UPGMA) clustering analysis. Overall, the germplasm resources of P. elliottii exhibited rich genetic diversity, with the majority of the genetic variation occurring within provenances. For the genetic improvement of high-resin-yielding slash pines, breeding programs should prioritize populations with high genetic diversity while carefully selecting superior individuals from within those populations. These findings provide a solid foundation for breeding high-resin-yielding varieties and for future research on the sustainable utilization of these valuable resources.

Evaluation of oxidative stress, biochemical parameters and in silico markers in different pea accessions in response to drought stress.

ARG6 and ARG10 pea accessions exhibited better tolerance to drought by keeping drought-associated attributes stable and higher, that is, stable chlorophyll content, high antioxidant activity, and the presence of polymorphic bands with stress-responsive EST-SSR markers. Each year, a significant portion of crops is lost due to various abiotic stresses, and even pea (Pisum sativum) crop growth and yield are severely affected by the challenges posed by drought stress. Drought is a critical factor that limits crop growth and development, and its impact is exacerbated by changes in the magnitude of climatic conditions. Drought induces oxidative stress in plants, leading to the accumulation of high concentrations of reactive oxygen species that damage cell structures and vital functioning of cells. The primary objective was to identify stress-tolerant plants by evaluating different morphological and biochemical attributes, such as biomass, chlorophyll content, relative water content, ascorbate peroxidase (APX), superoxide dismutase (SOD), and DPPH scavenging activity, as well as protein, proline, and phenolic content. Our study revealed that pea accessions (ARG6 and ARG10) were more resilient to drought stress as their chlorophyll, relative water, protein, and proline contents increased under drought conditions. Antioxidant enzymes, such as SOD, APX, and DPPH activities, also increased under drought stress in ARG10 and ARG6, suggesting that these accessions could bolster the antioxidant defense system in response to drought stress. Based on putative (cellular, biological, and metabolic) functions, ten EST-SSR primers were selected for the amplification study. Three EST-SSR primers, AUMP06_110, AUMP18_300, and AUMP31_250, were used for ARG6 and ARG10. Based on the correlation between the presence or absence of specific EST-SSR alleles, various physiological and morphological traits, and DPPH scavenging activity, both ARG10 and ARG6 demonstrated resistance to drought stress.

Development of SSR markers for genetic diversity analysis and species identification in Polygonatum odoratum (Mill.) Druce based on transcriptome sequences.

Polygonatum odoratum (Mill.) Druce is a well-known traditional Chinese herb belonging to the Polygonatum. However, the understanding of the genetic diversity of this species at the molecular level is limited due to the lack of transcriptomic and genomic information. In this study, 37,387 unigenes were assembled based on the transcriptome sequencing of the rhizome of Polygonatum odoratum (Mill.) Druce., and 11,021 single- sequence repeats (SSR) motifs, mainly consisting of single-nucleotide repeats (44.44%), dinucleotides (31.06%), and trinucleotides (22.59%), were identified. Based on these SSR motifs, 9,987 primer pairs of SSR markers were designed and 68 SSR markers were randomly selected for verification, of which 21 SSR markers showed polymorphisms among the 24 Polygonatum odoratum germplasms. Ninety-four alleles were detected: the observed alleles ranged from 2 to 11, the effective alleles varied from 1.086 8 to 4.916 8, the Shannon diversity index was 0.173 2~1.749 7, and the polymorphism information content PIC ranged from 0.076 7 to 0.803 9. Based on our analysis of genetic diversity (SSR genotypes) and population structure, we divided the 24 germplasm resources into two groups, indicating that the germplasm with similar geographical origins can be grouped together. In addition, the primers 'YZ14' and 'YZ47' could effectively distinguished the related species: Polygonatum kingianum Coll.et Hemsl., Polygonatum sibiricum Red., Polygonatum cyrtonema Hua, Polygonatum zanlanscianense Pamp. and Polygonatum odoratum (Mill.) Druce. This is the first study in which a dataset of expressed sequence tag (EST)-SSR markers is constructed for the Polygonatum odoratum (Mill.) Druce, and these newly developed EST-SSR markers provided a very efficient tool for genetic relationship analysis, species identification and marker-assisted selection breeding of Polygonatum odoratum (Mill.) Druce.

Population genetics analysis of Diospyrosmun A.Chev. ex Lecomte (Ebenaceae) based on EST-SSR markers derived from a novel transcriptome.

Diospyrosmun A.Chev. ex Lecomte (Ebenaceae), a native evergreen tree in Vietnam, has important economic and ecological values. The absence of effective and reliable molecular markers has hampered the study of D.mun's genetic diversity and population structure, even though it is an endemic and endangered species. Therefore, significant enrichment of genomic resources is urgently needed to uncover and better understand the genetic architecture of D.mun. This study aims to demonstrate an efficient and reliable tool to explore the polymorphism within D.mun germplasm. It provides a valuable platform for the breeding and conservation of this species and other endangered species worldwide. The Illumina HiSeq™ 4000 sequencing technology was applied for the transcriptomic analysis, genetic differentiation and population structure of D.mun in Vietnam. In this study, the transcriptomes of D.mun were analysed using the Illumina HiSeqTM 4000 sequencing system and a total of 5,588,615,700 base pairs were generated. De novo assembly indicated that 91,134 unigenes were generated (average length = 645.55 bp, N50 = 957 bp, Q20 = 98.08% and Q30 = 94.51%). A total of 92,798 and 21,134 unigenes had significant similarities amongst Nr and Swiss-Prot, respectively. In the GO database, 19,929 unigenes were annotated and these genes were divided into three major categories and 50 subcategories. In the KOG analysis, 18,499 unigenes were annotated and divided into 25 gene function categories. In the KEGG analysis, 12,017 unigenes were annotated. According to the related pathways involved, they could be classified into 56 subclasses. In this study, we have identified a total of 9,391 EST-SSR markers. Ten microsatellite loci were employed to assess the genetic diversity and structure of 82 adult D.mun trees across three populations in Vietnam. The results indicated moderate levels of genetic diversity with PIC = 0.77, NA = 3.9, NE = 2.8, Ho = 0.56 and HE = 0.58 and the fixation index value was recorded as positive for three populations (NS, NH and CP). Genetic differentiation among populations was low (FST = 0.045), suggesting limited gene flow (Nm = 5.34). This result indicates gene exchange between the populations of ancient D.mun from different geographical areas and regions. The analysis of molecular variance (AMOVA) showed that high genetic variation existed within individuals (91%) compared to amongst populations (4%). Genetic structure analysis, DAPC and the NJ tree indicated that the three populations were divided into three main clusters. With this study, we provide a molecular resoureces for the breeding and conservation of D.mun.

Exploration of Genetic Variation and Population Structure in Bergenia ciliata for its Conservation Implications.

Bergenia ciliata (Haw.)Sternb. is a perennial medicinal herb distributed in Indian Himalayan Region(IHR). A total of eight populations of B.ciliata were collected from diverse locales of IHR, and 17 EST-SSR markers were used in this study. The present study revealed moderate genetic diversity at the locus level with the mean number of alleles (Na = 7.823), mean number effective of alleles (Ne = 3.375), mean expected heterozygosity (He = 0.570), and meanShannon's diversity index (I = 1.264). The MSR (He = 0.543, I = 1.067) and DRJ populations (He = 0.309, I = 0.519) revealed the highest and lowest genetic diversity at the population level, respectively. AMOVA analysis showed that 81.76% of genetic variation was within populations, 10.55% was among populations, and 7.69% was among the regions. In addition, a moderate to high level of differentiation was found among the populations (FST = 0.182), which could be indicative of low to moderate gene flow (Nm = 0.669) in the B. ciliata populations. UPGMA and PCoA analysis revealed that eight populations could be differentiated into two groups, while the structure analysis of the 96 individuals differentiated into three groups. The Mantel test showed a positive relationship between genetic and geographical distance. The findings of this study will provide the development of conservation and germplasm management strategies for this valuable medicinal species.

Genetic diversity evaluation of Luculia yunnanensis, a vulnerable species endemic to Yunnan, Southwestern China based on morphological traits and EST-SSR markers.

Luculia yunnanensis is a vulnerable species endemic to Yunnan Province, Southwestern China, which has high ornamental value. Its wild population has not been fully protected and utilized for a long time, which is not conducive to the long-term stable development of this species. Genetic diversity assessment is the basis and prerequisite for the conservation of rare species. In this study, 21 phenotypic traits and 17 highly polymorphic EST-SSR markers were used to analyze the genetic diversity and genetic structure of 164 individuals from six L. yunnanensis populations. The coefficient of variation of 21 phenotypic traits ranged from 11.76% to 52.58% (mean=21.72%), and the coefficient of variation of 18 traits was less than 30%. The average values of Ne, I, Ho and He were 1.710, 0.619, 0.384, and 0.352, respectively. The genetic diversity of LLO (Ho = 0.476 and He = 0.426) and LCM (Ho = 0.424 and He = 0.381) populations in Lushui County was highest. The GDX populations (Ho = 0.335 and He = 0.269) isolated by Gaoligong Mountain had the lowest genetic diversity. The AMOVA results showed that 13.04% of the genetic variation was among populations and 86.96% was within populations. The average phenotypic differentiation coefficient of phenotypic traits among populations was 18.69%. The results of phenotypic and genetic variation analysis were consistent, indicating that the most of variation exists within population. Genetic structure, UPGMA clustering and PCA analysis results showed that the populations of L. yunnanensis had obvious geographical divisions, and the populations distributed in the southern region and distributed in the northern region of the Nujiang River clustered into one group respectively. Combining the results of phenotypic and molecular markers, we recommend that give priority to the protection of LLO, LCM and GDX population, in order to ensure the sustainable utilization of L. yunnanensis germplasm resources.

Phenotyping variability and detecting informative SSR and EST-SSR markers for malt and straw quality characteristics in a diverse panel of wild barley (H. spontaneum) under rainfed and irrigated conditions

Phenotyping variability and detecting informative SSR and EST-SSR markers for malt and straw quality characteristics in a diverse panel of wild barley (H. spontaneum) under rainfed and irrigated conditions

Characterisation of the Cinnamomumparthenoxylon (Jack) Meisn (Lauraceae) transcriptome using Illumina paired-end sequencing and EST-SSR markers development for population genetics.

Cinnamomumparthenoxylon is an endemic and endangered species with significant economic and ecological value in Vietnam. A better understanding of the genetic architecture of the species will be useful when planning management and conservation. We aimed to characterize the transcriptome of C.parthenoxylon, develop novel molecular markers, and assess the genetic variability of the species. First, transcriptome sequencing of five trees (C.parthenoxylon) based on root, leaf, and stem tissues was performed for functional annotation analysis and development of novel molecular markers. The transcriptomes of C.parthenoxylon were analyzed via an Illumina HiSeqTM 4000 sequencing system. A total of 27,363,199 bases were generated for C.parthenoxylon. De novo assembly indicated that a total of 160,435 unigenes were generated (average length = 548.954 bp). The 51,691 unigenes were compared against different databases, i.e. COG, GO, KEGG, KOG, Pfam, Swiss-Prot, and NR for functional annotation. Furthermore, a total of 12,849 EST-SSRs were identified. Of the 134 primer pairs, 54 were randomly selected for testing, with 15 successfully amplified across nine populations of C.parthenoxylon. We uncovered medium levels of genetic diversity (PIC = 0.52, Na = 3.29, Ne = 2.18, P = 94.07%, Ho = 0.56 and He = 0.47) within the studied populations. The molecular variance was 10% among populations and low genetic differentiation (Fst = 0.06) indicated low gene flow (Nm = 2.16). A reduction in the population size of C.parthenoxylon was detected using BOTTLENECK (VP population). The structure analysis suggested two optimal genetic clusters related to gene flow among the populations. Analysis of molecular variance (AMOVA) revealed higher genetic variation within populations (90%) than among populations (10%). The UPGMA approach and DAPC divided the nine populations into three main clusters. Our findings revealed a significant fraction of the transcriptome sequences and these newlydeveloped novel EST-SSR markers are a very efficient tool for germplasm evaluation, genetic diversity and molecular marker-assisted selection in C.parthenoxylon. This study provides comprehensive genetic resources for the breeding and conservation of different varieties of C.parthenoxylon.

EST-SSR marker development based on de novo transcriptomic assembly of Aesculus chinensis Bunge

EST-SSR marker development based on de novo transcriptomic assembly of Aesculus chinensis Bunge

Transcriptome analysis and development of EST-SSR markers in the mushroom Auricularia heimuer

Auricularia heimuer, the third most frequently cultivated edible mushroom species worldwide, has high medicinal value. However, a shortage of molecular marker hinders the efficiency and accuracy of genetic breeding efforts for A. heimuer. High-throughput transcriptome sequencing data are essential for gene discovery and molecular markers development. This study aimed to clarify the distribution of SSR loci across the A. heimuer transcriptome and to develop highly informative EST-SSR markers. These tools can be used for phylogenetic analysis, functional gene mining, and molecular marker-assisted breeding of A. heimuer. This study used Illumina high-throughput sequencing technology to obtain A. heimuer transcriptome data. The results revealed 37,538 unigenes in the A. heimuer transcriptome. Of these unigenes, 24,777 (66.01%) were annotated via comparison with the COG, Pfam, and NR databases. Overall, 2510 SSRs were identified from the unigenes, including 6 types of SSRs. The most abundant type of repeats were trinucleotides (1425, 56.77%), followed by mononucleotides (391, 15.58%) and dinucleotides (456, 18.17%). Primer pairs for 102 SSR loci were randomly designed for validity confirmation and polymorphism identification; this process yielded 53 polymorphic EST-SSR markers. Finally, 13 pairs of highly polymorphic EST-SSR primers were used to analyze the genetic diversity and population structure of 52 wild A. heimuer germplasms, revealing that the 52 germplasms could be divided into three categories. These results indicated that SSR loci were abundant in types, numbers, and frequencies, providing a potential basis for germplasm resource identification, genetic diversity analysis, and molecular marker-assisted breeding of A. heimuer.

Cloning a novel reduced-height (Rht) gene TaOSCA1.4 from a QTL in wheat.

Reducing plant height (PH) is one of the core contents of the "Green Revolution", which began in the 1960s in wheat. A number of 27 reduced-height (Rht) genes have been identified and a great number of quantitative trait loci (QTLs) for PH have been mapped on all 21 chromosomes. Nonetheless, only several genes regulated PH have been cloned. In this study, we found the interval of QTL QPh-1B included an EST-SSR marker swes1079. According to the sequence of swes1079, we cloned the TaOSCA1.4 gene. We developed a CAPS marker to analyze the variation across a natural population. The result showed that the PH was significantly different between the two haplotypes of TaOSCA1.4-1B under most of the 12 environments and the average values of irrigation and rainfed conditions. This result further demonstrated that TaOSCA1.4 was associated with PH. Then, we validated the TaOSCA1.4 via RNAi technology. The average PHs of the wild-type (WT), RNAi lines 1 (Ri-1) and 2 (Ri-2) were 94.6, 83.6 and 79.2 cm, respectively, with significant differences between the WT and Ri-1 and Ri-2. This result indicated that the TaOSCA1.4 gene controls PH. TaOSCA1.4 is a constitutively expressed gene and its protein localizes to the cell membrane. TaOSCA1.4 gene is a member of the OSCA gene family, which regulates intracellular Ca2+ concentration. We hypothesized that knock down mutants of TaOSCA1.4 gene reduced regulatory ability of Ca2+, thus reducing the PH. Furthermore, the cell lengths of the knock down mutants are not significantly different than that of WT. We speculate that TaOSCA1.4 gene is not directly associated with gibberellin (GA), which should be a novel mechanism for a wheat Rht gene.

Gene flow from Fraxinus cultivars into natural stands of Fraxinus pennsylvanica occurs range-wide, is regionally extensive, and is associated with a loss of allele richness.

In North America, a comparatively small number of Fraxinus (ash) cultivars were planted in large numbers in both urban and rural environments across the entire range of Fraxinus pennsylvanica Marsh (green ash) over the last 80 years. Undetected cultivar gene flow, if extensive, could significantly lower genetic diversity within populations, suppress differentiation between populations, generate interspecific admixture not driven by long-standing natural processes, and affect the impact of abiotic and biotic threats. In this investigation we generated the first range-wide genetic assessment of F. pennsylvanica to detect the extent of cultivar gene flow into natural stands. We used 16 EST-SSR markers to genotype 48 naturally regenerated populations of F. pennsylvanica distributed across the native range (1291 trees), 19 F. pennsylvanica cultivars, and one F. americana L. (white ash) cultivar to detect cultivar propagule dispersal into these populations. We detected first generation cultivar parentage with high confidence in 171 individuals in 34 of the 48 populations and extensive cultivar parentage (23-50%) in eight populations. The incidence of cultivar parentage was negatively associated with allele richness (R2 = 0.151, p = 0.006). The evidence for a locally high frequency of cultivar propagule dispersal and the interspecific admixture in eastern populations will inform Fraxinus gene pool conservation strategies and guide the selection of individuals for breeding programs focused on increasing resistance to the emerald ash borer (Agrilus planipennis Fairmaire), an existential threat to the Fraxinus species of North America.